WO1997026911A1 - Vaccins contre la malaria faisant appel a un peptide de msa1 - Google Patents

Vaccins contre la malaria faisant appel a un peptide de msa1 Download PDF

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Publication number
WO1997026911A1
WO1997026911A1 PCT/US1997/001395 US9701395W WO9726911A1 WO 1997026911 A1 WO1997026911 A1 WO 1997026911A1 US 9701395 W US9701395 W US 9701395W WO 9726911 A1 WO9726911 A1 WO 9726911A1
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Prior art keywords
peptide
msal
sequence
anchor
msalc
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PCT/US1997/001395
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English (en)
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Eugene A. Davidson
Shutong Yang
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Georgetown University
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Priority to AU22490/97A priority Critical patent/AU2249097A/en
Priority to JP9527100A priority patent/JP2000504223A/ja
Priority to EP97905652A priority patent/EP0885012A4/fr
Priority to CA002245002A priority patent/CA2245002A1/fr
Publication of WO1997026911A1 publication Critical patent/WO1997026911A1/fr
Priority to US09/117,415 priority patent/US6551586B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/788—Of specified organic or carbon-based composition
    • Y10S977/802—Virus-based particle
    • Y10S977/803—Containing biological material in its interior
    • Y10S977/804—Containing nucleic acid

Definitions

  • the present invention relates to novel DNA constructs comprising a vector linked to a DNA segment which encodes a protein containing a signal protein at its N-terminus and an anchor sequence at its C-terminus.
  • the present invention relates to vaccines which are useful for the prevention and treatment of malaria caused by Plasmodium falciparum in humans.
  • Plasmodium vivax Plasmodium malariae. Plasmodium falciparum and Plasmodium ovale. Of these, Plasmodium falciparum produces the most pathogenic of the malarias and often results in death. It is responsible for about half of the human cases of malaria found worldwide.
  • the life cycle of the malaria parasite provides several stages at which interference could lead to cessation of the infective process. Included among these stages is the invasion of the erythrocyte by the merozoite.
  • the merozoite represents a potentially attractive target (and perhaps the only target) from which a vaccine may be produced, because the free merozoite, although it has a limited lifetime (one to two hours) occurs earlier in the life cycle of malaria, and the emergence of later stage sexual forms, which are responsible for transmission of the disease, depends upon the erythrocytic stage.
  • the general life cycle of malaria parasites is the same for human and other animal malaria parasites, thus allow ⁇ ing model studies to be conducted on a rodent species with accurate translation to the human parasite.
  • the rodent malaria parasite strain Plasmodium ber ⁇ rhei Anka has a pathology very similar to the FCR-3 strain of Plasmodium fal ⁇ ciparum (a well studied variant of the human parasite) .
  • the blood stage of the human parasite can be grown in the laboratory (in human red cells) thus affording a system for studying the effects of antibodies/inhibitors on the inva ⁇ sion process, and the erythrocytic phase.
  • a human becomes infected with malaria from the bite of a female Anopheles mosquito.
  • the mosquito inserts its probe into a host and in so doing, injects a sporozoite form of Plasmodium falciparum. present in the saliva of the mosquito.
  • the sporozoites which have been injected into the human host are cleared into a number of host tissue cells, including liver parenchyma cells (hepatocytes) and macro ⁇ phages. This phase is known as the exoerythrocytic cycle because at this point in the life cycle the organism has not yet entered red blood cells.
  • hepatocytes liver parenchyma cells
  • macro ⁇ phages macro ⁇ phages.
  • This phase is known as the exoerythrocytic cycle because at this point in the life cycle the organism has not yet entered red blood cells.
  • sporozoites undergo a transformation into trophozoites, which incubate and undergo schizogony, rupture and liberate tissue merozoites. This process takes approximately 7-10 days and, depending upon species, may repeat itself several times, dur ⁇ ing which time the host feels no effects. In Plasmodium fal ⁇ ciparum. this repetition does not occur.
  • the liver or other tissue cells burst open and release numerous merozoites into the bloodstream.
  • red blood cells where they enter the erythrocytic phase of the life cycle.
  • young plasmodia have a red nucleus and a ring-shaped, blue cytoplasm.
  • the plasmodium divides into merozoites, which may break out of the red blood cell, enter other erythrocytes and repeat the multiplication process. This period lasts approximately 48 hours.
  • the gametocytes are capable of mating only in the mosquito.
  • a mosquito in order for Plasmodium falciparium to pro ⁇ quiz sporozoites for infecting a second human host, a mosquito must first bite a human host carrying gametocytes. These gametocytes mature into macrogametes, mate in the mosquito's stomach and produce a zygote.
  • the zygote (ookinete) is active and moves through the stomach or the midgut wall. Under the lining of the gut, the ookinete becomes rounded and forms a cyst called an oocyst, in which hundreds of sporozoites develop.
  • Sporozoites thereafter invade the entire mosquito and many of them enter the salivary glands where they are in a favorable position to infect the next host when the mosquito feeds on its blood. The life cycle thereafter simply repeats itself in another human host.
  • live vaccinia virus was used as a vaccine to eradicate smallpox successfully, and a recombinant vaccinia virus expressing viral antigens has been shown to induce a strong antibody response in immunized animals, conferring pro ⁇ tection against disease (Arita, I., Nature. 1979, 279, 293- 298) . Furthermore, it has been shown in animal models that co-presentation of potential immunogens with highly immunogenic vaccinia virus proteins can elicit a strong immune response against that specific immunogen (Moss and Flexner, Annals of the New York Academy of Sciences, 86-103; Mackett and Smith, J. Gen. Virol..
  • a panel of transfer vectors have been constructed that allow insertion of foreign genes into several sites within the 180kb vaccinia virus genome (Earl and Moss, Current Protocols in Molecular Biology. 1993, 16.17.1-16.17.16) . Also, it has been reported that >25kb of foreign DNA can be inserted into the vaccinia virus genome (Smith and Moss, Gene, 1983, 25, 21-28) .
  • the correct processing Chokrabarta, et al., Nature. 1986, 320, 535-537) and the appropriate post-translational modification (Hu, et al., Nature. 1986, 320, 537-540; Ball, et al., Proc. Natl. Acad. Sci. USA.
  • vaccinia virus vaccine has the advantage of being relatively inexpensive and easily stored, transported and delivered, features which are particularly important in the developing countries where malaria is most prevalent.
  • Proteins on the surface of merozoites are good targets for an immune response and are good malaria vaccine candidates because merozoites are the only stage in the asexual blood cycle in which the parasite is exposed to the immune system.
  • the 190kD glycoprotein of Plasmodium falciparum, precursor to major merozoite surface antigenl (MSAl) which is synthesized during schizogony, is considered a promising candidate for a blood-stage malaria vaccine (Black an, et al., Mol. Biochem. Parasitol.. 1991, 49, 29-34; Perrin, et al., J. Exp. Med.. 1984, 160, 441-451; Siddiqui, et al., Proc. natl.
  • the high-molecular weight precursor is processed into 88kD, 30kD, 38kD and 42kD fragments which remain as complexes on the merozoite surface (Holder, et al., Parasitology. 1987, 94, 199-208; McBride and Heidrich, Mol. Biochem. Parsitol. , 1987, 23, 71-84) .
  • the complex is released from the membrane by cleavage of the 42kD anchor fragment, and a 19kD carboxyl-terminal fragment remains on the merozoite membrane and is carried into the invaded erythrocytes (Black- man, et al., supra; Blackman, et al., Mol. Biochem. Parasitol.. 1991, 49, 35-44) .
  • the complete MSAl of unprocessed P. falciparum has been used to provide partial or complete protection against challenge infection (Blackman, et al., Mol. Biochem. Parasitol., 1991, 49, 29-34; Perrin, et al., J. EXP. Med..
  • mAbs monoclonal antibodies directed against glycophorin A, the putative erythrocyte receptor for £_;_ falciparum were prepared.
  • One of these mAbs, designated 2B10 is capable of blocking the binding of MSAl to human erythrocytes and inhibiting the invasion of P. falciparum merozoites into human erythrocytes (Su, et al. Infect. lmm.. 1993, 151, 2309) .
  • the anti-idiotype antibody of 2B10 recognized the C-terminal (1047-1640aa) region of MSAl in a western blot (Su, et al., J. Immunol.. 1995) and appears to recognize the same site on glycophorin A as the merozoite.
  • the present invention relates to a malaria vaccine comprising an expression vector, preferably, a vaccinia virus system which expresses a protein corresponding substantially to a specific domain of the major merozoite surface antigen 1 (MSAl) of Plasmodium falciparum or an immugenic peptide por ⁇ tion thereof.
  • MSAl major merozoite surface antigen 1
  • the DNA coding for the MSAl protein domain is expressed by the vac ⁇ cinia virus after administration to a patient.
  • the MSAl protein or sub-fragment which is then expressed in the patient raises a humoral and/or cell-mediated response to the merozoite malaria antigen, which response provides the effect of protecting the vaccinated patient from a subsequent malaria infection.
  • the vaccinia virus system continues to express antigen in the patient for a period of days, months or even years, thus reinforcing the immunogenic response of the patient to the expressed antigen.
  • the MSAl peptide antigen or immunogenic peptide por ⁇ tion thereof which is expressed by the expression vector vac ⁇ cinia virus may also comprise a signal peptide and/or an anchor peptide sequence. It has been found that the addition of a signal and/or anchor peptide to the expressed MSAl antigen in vaccines according to the present invention unex ⁇ pectedly enhances the immunogenicity to the patient of the MSAl protein of Plasmodium falciparum.
  • the inclusion of a signal and/or anchor protein with MSAl can be expressed by a vaccinia virus system accord ⁇ ing to the present invention and the expressed peptide will produce a significantly greater immunogenic response than the MSAl peptide alone or in combination with an adjuvant. It is also an unexpected result that the inclusion of a signal and anchor sequence in the MSAl peptide sequence expressed by the vaccinia virus will produce an immunogenic response which may be as much as 100 fold greater than the immnogenic response which is produced by the MSAl peptide which does not contain a signal or anchor peptide sequence.
  • Methods of inducing an immunogenic response in a patient are also contemplated by the present invention.
  • a patient is administered an amount of a vaccinia virus capable of expressing the MSAl peptide of Plasmodium falciparum such that the patient develops an immunogenic response to the expressed peptide.
  • the immunogenic response generated preferably will be "substantially protective", i.e., will protect the patient from some of the more severe symptoms and physiological states of the malaria disease, including the death of the patient from malaria.
  • the present invention also relates to an immunogenic dosage form as a vaccine, for inducing an immunogenic response to the merozoite stage in the life cycle of Plasmodium fal ⁇ ciparum.
  • Methods of vaccinating a patient against a malaria infection are also contemplated by the present invention.
  • a patient is vaccinated against a Plasmodium fal ⁇ ciparum infection by administering an immunogenic response producing effective amount of a vaccinia virus capable of expressing the MSAl peptide or an immunogenic peptide portion thereof of Plasmodium falciparum in the patient.
  • the present invention also relates to chimeric proteins or peptides comprising the peptide sequence cor ⁇ responding to the major merozoite surface antigen 1 (MSAl) of Plasmodium falciparum or an immugenic peptide portion thereof in combination with a signal sequence and/or anchor sequence, more preferably both a signal sequence and an anchor sequence.
  • MSAl major merozoite surface antigen 1
  • Figure l is a diagrammatic representation of the con ⁇ struction of recombinant vaccinia viruses incorporating the sequences corresponding to MSAlC-(Si,A) , MSAlC-(Si,nA) , MSA1C- (nSi,A) and MSAlC-(nSi,nA) .
  • Figures 2-5 represent the gene sequences for MSA1C- (Si,A) , MSAlC-(Si,nA) , MSA1C-(nSi,A) and MSA1C-(nSi ,nA) .
  • Figure 6 is a diagrammatic representation of four recombinant vaccinia viruses of the present invention: rv- MSAl-(Si,A); rv-MSAl-(Si,nA) ; rv-MSAl-(nSi,A) ; rv-MSAl- (nSi,nA) .
  • This figure shows the schematic of the genome of recombinant vaccinia viruses expressing the different MSAl constructs.
  • Tki and TK R right and left regions ofthe vac ⁇ cinia virsu thymidine kinase gene; LacZ, beta-galacosidase gene.
  • Si signal region of MSAl
  • nSi no signal region of MSAl
  • A anchor region of MSAal
  • nA no anchor region.
  • Figure 7 is a diagrammatic representation of the results obtained from Western blot analysis of recombinant vaccinia virus expressed proteins using anti-MSAlC-A mouse serum as a probe, of proteins expressed from BSC-1 cells infected with vaccinia virus.
  • A pellet of rV.V-MSAlC(Si,nA) infected cells;
  • B supernatant of rV.V-MSAlC(Si,nA) infected cells;
  • C pellet of rV.V-MSAlC(Si,A) infected cells;
  • D supernatant of rV.V-MSAlC(Si,A) infected cells;
  • E supernatant of rV.V-MSAlC(Si,A) infected cells;
  • pellet of rV.V-MSAlC(nSi,nA) infected cells F. supernatant of rV.V- MSAlC(nSi,nA) infected cells; G. pellet of rV.V-MSAlC(nSi,A) infected cells; H. supernatant of of rV.V-MSAlC(nSi,A) infected cells; I. pellet of WR infected cells; J. super ⁇ natant of WR infected cells.
  • Figure 8 is a diagrammatic representation of results obtained from immunofluorescence staining of non-permeabilized infected cells.
  • HeLa cells seeded on pre-treated coverslips for 48 hours were infected with recombinant and wild-type vac ⁇ cinia virus at an M.O.I, of 5.
  • the cells were probed with anti-MSAlC-A serum and laveled with FITC goat anti-mouse.
  • A,B rV.V-MSAlC(Si,nA) infected cells
  • C,D rV.V-MSAlC(Si,A) infected cells
  • E,F rV.V-MSAlC(nSi,nA) infected cells
  • G,H rV.V-MSAlC(nSi,A) infected cells
  • I,J WR infected cells.
  • Figure 9 is a diagrammatic representation of results obtained in rabbits immunized intradermally with the recom ⁇ binant vaccinia viruses of the present invention.
  • Five rab ⁇ bits were immunized intradermally with recombinant and wild- type vaccinia virus, and one rabbit was immunized intravenously with rV-MSAlC-(Si,A) .
  • the I.D. immunizations occurred on days 0, 21, 47 and 68, and the I.V. immunizations occurred on days 0, 47 an 68.
  • Blood samples taken on days 33, 54, and 78 were analyzed for antibodies to the trpE-MSAlC-A protein (C-terminus of MSAl expressed and purified from E.
  • FIG 10 is a diagrammatic representation of results obtained in Balb/c mice which were inoculated with vaccinia viruses of the present invention.
  • Balb/c mice were inoculated I.P. with recombinant and wild-type vaccinia virus on days 0, 21, 42 and 63.
  • Blood samples taken on days 11, 31, 51, 73 93, 123, 147, 167 and 187 were analyzed for antibodies to trpE- MSA1C-A protein (C-terminus of MSAl expressed and purified from E_j_ coli) using ELISA.
  • Sera were analyzed in duplicate: (J " ) rV-MSAlC-(Si,A) ; (o) (rV-MSAlC-(Si,nA) ; (X) (rV-MSAlC- (nSi,A); (O) (rV-MSAlC-(nSi,nA) ; (+) WR.
  • Figure 11 is a diagrammatic representation of the results obtained from a Western blot analysis of the recogni ⁇ tion of a 190kD of Pj. falciparum by vaccinia virus induced mouse antibodies.
  • Schizont stage parasites were lysed by boiling in sample buffer and loaded onto a 4-20% Tris-glycine gradient gel, and the proteins were transferred to a PVDF mem ⁇ brane by electrophoresis. These blots were then probed with anti-MSAlC-(Si,A) , anti-MSAlC-A or anti-WR antibodies and labelled with phasphatase-conjugated goat anti-mouse IgG.
  • chimeric protein is used to describe the non- natural peptide sequences according to the present invention which comprise the expressed protein or peptide and an anchor peptide and/or a signal peptide.
  • chimeric peptides generally are synthetic peptides pro- prised by an expression vector which contain a desired target protein or peptide (either MSAl or an immunogenic peptide por ⁇ tion thereof) in combination with another peptide sequence (either an anchor or signal peptide sequence) .
  • the term "patient” is used to describe an animal, including a mammal and especially including a human patient which is administered a dosage form of an expression vector or chimeric protein according to the present invention.
  • the expression vector encodes for MSAl or an immunogenic peptide portion therof and expresses the encoded protein or peptide in the patient.
  • expression vector is used to describe the means by which nucleic acid, including DNA, cDNA, RNA or variants thereof, more preferably DNA fragments, encoding for a specific peptide or protein, may be introduced into the patient or the patient's tissue in order to express or produce the desired protein.
  • Such vectors include any vectors into which a nucleic acid sequence encoding for the desired MSAl protein or immunogenic peptide fragment thereof, anchor pep ⁇ tide sequence and/or, signal protein sequence may be inserted (along with any required or optional operational elements) into a host organism and replicated.
  • Expression vectors may also be used to simply produce chimeric peptide in culture for isolation.
  • Preferred vectors are those which are capable of expressing the peptide or protein sequences in mammalian cells and whose restriction sites are well known and which contain the required operational elements for expression of the desired protein or peptide sequence.
  • the vector is preferably a vaccinia virus vector, adenovirus vector or herpes virus vector which has the capa ⁇ city to infect a mammalian cell and express or synthesize proteins utilizing the host's biosynthetic mechanism.
  • the viral vector used for delivery should optimally be one which infects cells but which does not cause lysis due to replication (i.e., an attenuated or partially disabled virus selected from among adenovirus, vaccinia virus and herpes viruses, among similar types).
  • the vector will infect the host cells and, using the host cells' biosynthetic pathways, synthesize encoded protein or peptide fragment.
  • Any immunizing vehicle which has a detailed genetic and human use history may be used as the expression vector in the present invention.
  • the preferred expression vector is a viral vector, more preferably, a vac ⁇ cinia virus vector, for example, as described by Earl and Moss, Current Protocols in Molecular Biology. 1993, 16.17.1- 16.17.16) and Smith and Moss, Gene, 1983, 25, 21-28.
  • any vaccinia or other viral vector which may be used in the above-described manner may be appropriate for use in the pres ⁇ ent invention.
  • the expression vector should contain at least one promoter, at least one operator, at least one terminator codon, and any other sequences which are necessary for the efficient transcription and subsequent translation of the nucleic acid from the vector.
  • the expression vectors will advantageously comprise at least one origin of replication which is recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid (preferably, DNA) sequence.
  • vaccines comprise an expression vector, preferably a vaccinia virus system which expresses an antigenic protein after administration of the vaccinia virus system to an animal, such as a mammal.
  • Vac ⁇ cines may also comprise chimeric peptides comprising MSAl or an immunogenic peptide portion thereof in combination with a signal peptide sequence and/or an anchor peptide sequence.
  • the method of administering the vaccine(s) according to the present invention may vary and include intravenous, buccal, oral, transdermal and nasal, among others, but intramuscular or subcutaneous administration is the most common method of administration.
  • MSAl protein or "MSAl peptide” is used to describe the major merozoite surface antigen 1 of the merozoite stage of Plasmodium falciparum or immunogenic pep ⁇ tide portions thereof.
  • MSAl is the major surface protein of the merozoite stage of Plasmodium falciparum. It is a 190kD glycoprotein which is synthesized during schizogony life-cycle stage. The high molecular weight precursor is processed into 88kD, 30kD, 38kD and 42kD fragments which remain as complexes on the merozoite surfaces.
  • the complex is released from the merozoite membrane by cleavage of the 42kD anchor fragments and a l9kD carboxy-terminal fragment remains on the merozoite membrane and is carried into the invaded erythrocytes.
  • the complete MSAl gene sequence is available on the data base UNDP/WORLDBANK/WHO/TDR Malaria Sequence.
  • the preferred por ⁇ tion of MSAl for expression in the vaccinia virus system according to the present invention is a carboxy-terminal region (corresponding to amino acids 1047 to 1640) of MSAl.
  • the expressed protein may be MSAl or any portion thereof, preferably such that the MSAl portion contains at least the carboxy-terminal region of MSAl.
  • carboxy-terminal region of MSAl and “carboxy- terminal MSAl peptide” are used to describe that portion of the MSAl protein corresponding to amino acids 1047 to 1640 which is the preferred expressed antigen peptide sequence in the present vaccines. It represents a preferred target for the development of humoral and/or cell mediated response because of the degree of specificity of the immune response which can be elicited against such a protein segment.
  • the expression of the C-terminal MSAl peptide according to the instant invention produces a specific immune reactivity which becomes less specific as more of the MSAl protein is incorporated into the vaccinia virus system.
  • the above terms to d ⁇ escribe the C-terminal MSAl peptide include not only the 593 amino acid peptide referred to above, but any peptide substan ⁇ tially corresponding to that 593 amino acid peptide.
  • Any expressed peptide which substantially corresponds to the MSAl protein or an immunogenic peptide portion of the MSAl protein and, preferably, also contains a peptide cor ⁇ responding to at least an immunogenic portion of the C- ter inal MSAl peptide, may be used in the present vaccines.
  • expressed peptides corresponding to MSAl protein and/or an immunogenic peptide portion thereof in combination with a signal sequence or anchor sequence may also be used in the present invention.
  • signal peptide "signal sequence” or “signal protein” is used to describe a 7-30 unit amino acid peptide sequence, preferably about a 15-26 unit amino acid peptide sequence, which is generally found at or near the N-terminus of the expressed protein or peptide which is used in the pres ⁇ ent invention in order to substantially enhance the biological activity of the protein or peptide expressed in the patient according to the present invention.
  • Signal sequences generally contain hydrophobic peptide sequences of between about 7 and 30 amino acid units, more preferably, about 15 to 26 amino acid units, even more preferably about 16 to 24 amino acid units and most preferably about 18 to 20 amino acids units appear to be essential for the targeting of protein chains (generally, secretory proteins) to membranes within the cell.
  • hydrophobic sequences are of sufficient length to cross the lipid bilayer of the cell membranes.
  • Signal sequences serve as organizers for the cellular traffic of mac ⁇ romolecules. These proteins are believed to play a central role in the translocation of polypeptide chains across mem ⁇ branes.
  • the incorporation of a sig ⁇ nal protein sequence at the amino terminus of the protein or peptide sequence expressed by the vaccinated patient is asso ⁇ ciated with the substantial enhancement in the biological activity (including the therapeutic effect of immunogenicity) associated with the expressed protein or peptide.
  • signal sequences which are known in the art may be used in the present invention.
  • yeast or lower trophic order signal sequences clearly mammalian signal sequences are preferred for use in mammals and the specific species signal sequences are most preferred for use in the desired mammalian species to be treated.
  • a human signal sequence is most preferred.
  • Signal sequences for use in the present invention generally contain three regions, a first or c region at the carboxy end of the peptide (which serves as the cleavage site for a signal peptidase enzyyme) , comprising about 5 to 7 amino acid residues which tend to be highly polar but uncharged; a second or h region which is N-terminal to the c region, generally about 7 to 13 amino acid residues in length and highly hydrophobic (comprised primarily of Leu, Ala, Met, Val, lie, Phe, and Trp amino acids, but may contain an occasional Pro, Gly, Ser or Thr amino acid residue) ; and a third region or n-region of highly variable length and composition, but generally carrying a net positive charge contributed by the N- terminus (negative charges contributed from acidic residues are also known) and any charged residues.
  • c region and the h region are between 1 and 3 amino acid residues which tend to be small and uncharged (Ala, Gly, Ser, others) .
  • Synthetic homopolymeric h regions comprised of amino acids selected from the group consisting of leucine, isoleucine, phenylalanine, valine, alanine and tryptophan, preferably leucine, isoleucine and phenylalanine may be used in the signal proteins according to the present invention. See generally, von Heijne, European Journal of Biochemistry. (1983) , 133, pp. 17-21.
  • the signal sequences which are used in the present invention preferably encompass eukaryotic signal sequences, preferably between 7 and 30 amino acid units in length, preferably between 15 and 26 units, more preferably between about 16 and 26 amino acids, even more preferably between 18 and 20 amino acid units.
  • the c region of the signal peptide should be more polar and the boundary between the h and c regions between residues -5 and - 6, or -7 or -8 (counting from the position of cleavage of the signal sequence- i.e., the first amino acid of the mature or expressed protein or peptide is +1) is between 1 and 3 amino acid residues which tend to be small and uncharged (Ala, Gly, Ser, others) .
  • Position preferences in the h/c for amino acids are as follows:
  • -10 most preferably leucine or alternatively, isoleucine, valine, alanine, or phenylalanine;
  • leucine most preferably leucine, alternatively, isoleucine, alanine, valine, phenylalanine;
  • leucine most preferably leucine, alternatively isoleucine, alanine, valine, glycine, phenylalanine;
  • -7 most preferably alanine, alternatively, leucine, isoleucine, valine, phenylalanine;
  • -6 most preferably valine, alternatively leucine, valine, isoleucine, phenylalanine, alanine;
  • -5 most preferably proline, alternatively glycine, alanine, leucine, valine;
  • -4 most preferably glycine, alternatively proline, leucine, alanine, valine;
  • leucine alternatively phenylalanine
  • -1 most preferably alanine, alternatively glycine.
  • the h region may vary in length as well.
  • the n region is polar, contains positively charged amino acids (predominantly lysine and arginine) and varies with the overall length of the signal peptide as described above.
  • the c region extends from residues -1 to -5 of the signal peptide/expressed or mature protein.
  • the c region is N-terminus to the expressed or mature protein
  • the h region is N-terminus to c region (with a 1-3 amino acid boundary between the c and h region)
  • the n region is a positively charged N-terminus to the h region.
  • the n region is variable in length and generally positively charged (with a preferred charge of +2)
  • the h region is hydrophobic and variable in length and the c region preferably contains about five (5-7) generally polar amino acids.
  • the end of the hydrophobic domain (i.e., the boundary between the hydrophobic residues enumerated above) should preferably be at positions -6/-5.
  • the signal sequence should comprise a 5 to 10 unit residue initial sequence (beginning with methionine) followed by at least a seven residue sequence (as described above) and an additional amino acids from 1 to 10 residues in length.
  • a typical sequence for the region noted about is: ILLLLAV .
  • the signal sequence used should be characteristic of the cell type used for expression of the protein. Thus, in veterinary applications, the signal sequence most preferably used should be that of the animal to be treated. Often a sig ⁇ nal sequence which is mammalian in character is acceptable. Most mammalian signal sequences will have significant efficacy in expressing proteins or peptides in other mammalian cells. Human signal sequences are preferably used for human applica ⁇ tions.
  • amino acid sequences corresponding to the above- described signal peptide DNA sequences are:
  • anchor protein or “anchor peptide sequence” is used to describe proteins or peptides which are anchored to the external surface of the plasma membrane generally by covalent bonding to glycans containing phosphatidyl inositol. These structures to which the anchor protein or peptide is bonded are often referred to as glycosyl phosphatidylinositols or GPIs. In all cells, anchor proteins covalently bonded to GPIs are found on the external face of the plasma membrane of cells or on the lumenal surface of secretory vesicles.
  • an “anchor protein” or “anchor peptide” comprises a peptide sequence preferably of about 15-35 residues in length which is generally expressed at the carboxy-terminus of the protein or peptide expressed by the expression vector according to the present invention (3' end of the DNA sequence expressing the desired protein or pep ⁇ tide and carboxyl terminus of the expressed protein or pep ⁇ tide) .
  • many of the proteins or pep ⁇ tides which are expressed in the patient and in particular, the immunogenic proteins or peptides of vaccines according to the present invention which are expressed in the patients pro ⁇ shall a biological or immunogenic response in the patient which is substantially enhanced when an anchor peptide is incor ⁇ porated at the carboxy terminus of that protein or peptide.
  • the inclusion of a signal protein at or in the proximity of the N-terminus, in addition to the anchor peptide at the carboxy-terminus of the expressed protein is associated with an unexpected enhancement in the biological effects of the expressed protein. This is especially true where the expressed protein is antigenic or immunogenic in nature.
  • the carboxy-terminus of the expressed protein or pep ⁇ tide residue is modified by attachment of a glycolipid anchor, which serves to anchor the modified protein or peptide to the cell surface.
  • the peptide residue to which the GPI anchor is added is always one of small amino acids, such as glycine, aspartic acid, asparagine, alanine, serine and cysteine. These occur at the carboxyl terminus of the protein/peptide of interest and thus can be specified by inclusion of the appropriate codons in the DNA fragment to be added to the cDNA sequence specifying the protein/peptide of interest. In addi ⁇ tion, the two residues downstream of the anchor addition site are usually small.
  • the cleavage/anchor addition site resides in a domain of three small amino acid residues, although the central of the three residues has less stringent steric requirements.
  • additional amino acids preferably, polar ones such as lysine or arginine as well as threonine, alanine and proline
  • the remainder of the addition signal sequence will contain from 15 to 35 amino acids with a hydrophobic domain at the extreme carboxyl terminus.
  • This domain should extend for 15- 25 amino acids and will include amino acids such as valine, leucine, isoleucine, alanine, pphenylalanine, but may also contain proline and glycine as well as tryptophan.
  • a typical such sequence is as follows:
  • the small sequence is in bold face with the left por ⁇ tion represeting the terminus of the protein and the D residue the site of GPI addition.
  • the right hand portion is that cleaved during GPI addition with the underlined sequence indicating the hydrophobic terminus.
  • the anchor peptide may have a cleavable N-terminal sequence, which directs the peptide to the endoplasmic reticulum and the cellular trafficking pathway where the GPI anchor is added.
  • the anchor peptide also has a predominantly hydrophobic sequence at the extreme carboxy terminus which generally ranges in size from about 15 to about 35, more preferably about 15 to 30, and even more preferably about 15 to 25 amino acid residues, signals the addition of the GPI anchor and is cleaved off concurrent with GPI addition. It is the hydrophobicity rather than the sequence itself which is important for anchor addition.
  • Anchor addition is generally a transamidation reaction in which the free ethanolamine amino group of the GPI precursor attacks (by way of nucleophilic addition) a peptide bond at the target amino acid, which becomes the C-terminal amino acid.
  • anchor addition site an amino acid residue within this hydrophilic spacer selected from the group consisting of glycine, aspartic acid, arginine, asparagine, alanine, serine and cysteine.
  • the two residues downstream from the anchor addition site are also usually small amino acid residues apparently to minimize steric hindrance at the anchor addition site.
  • the GPI portion is preassembled and added as a single unit to a specific amino acid residue near the carboyxl terminus of the expressed protein or peptide.
  • the carboxyl terminal region may be characterized by the presence of a C-terminal signal peptide whichis preferably ten to thirty amino acids in length and provides the information needed to add the GPI anchor.
  • the actual amino acid residue to which the GPI structure is attached is called the omega site and this residue should be glycine, alanine, cysteine, serine, asparagine or aspartic acid.
  • the omega +1 site (towards the carboxyl terminus of the expressed, unprocessed protein) preferably is selected from glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate and threonine.
  • the omega +2 site is alanine or glycine.
  • the omega +2 site is followed by a hinge or spacer of ideally 5 to 7 amino acids that preferably contains charged amino acids and proline; this is followed in turn by a preferably hydrophobic sequence of amino acids which terminate the carboxyl signal peptide.
  • the overall structure of the anchor peptide may be summarized as a 15-35 amino acid peptide at the carboxyl terminus of the expressed protein or peptide.
  • This anchor peptide sequence (reading from the terminus towards the amino end) begins with a hydrophobic stretch of amino acids of vari ⁇ able length, followed by a sequence of preferably 5-7 amino acids which contains charged residues, followed by three amino acids (either glycine or alanine at the omega +2 site) ; any of glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate and threonine at the +1 omega site; and any of glycine, alanine, serine, cysteine, aspartic acid or asparagine at the omega site.
  • the signal peptide sequence is generally found at the N-terminus (directly at the N-terminus or removed as much as 1,000 or more amino acids from the N-terminus) and the anchor peptide sequence is generally found at the carboxy-terminus of the expressed protein or peptide
  • the signal peptide may be found at or near the carboxy terminus of the expressed target protein or peptide.
  • anchor sequences which are known in the art may be used in the present invention.
  • yeast or lower trophic order anchor sequences clearly mammalian anchor sequences are preferred for use in mammals and the specific species signal sequences are most preferred for use in the desired mammalian species to be treated.
  • a human anchor sequence is most preferred.
  • anchor peptide DNA sequence is preferably used:
  • the amino acid sequence corresponding to the above- described anchor peptide DNA sequence is: FLGISFLLILMLILYSFI.
  • the signal peptide sequence is generally found at the N-terminus (directly at the N-terminus or somewhat removed- by as much as 1,000 or more amino acids from the N-terminus) and the anchor peptide sequence is generally found at or in the proximity of the carboxy-terminus of the expressed protein or peptide
  • the signal peptide may be found at or near the carboxy terminus and the anchor peptide may be found at or near the N-terminus of the expressed target protein or peptide.
  • an effective amount refers to an amount or concentration of recombinant vaccinia virus effective to pro ⁇ prise a protective immune or therapeutic response with respect to the disease malaria.
  • an effective amount of vaccinia virus which is administered to a human patient will vary depending upon a number of factors associated with that patient, including whether the patient previously has been exposed to Plasmodium falciparum before.
  • An effective amount of vaccinia virus can be determined by varying the dosage of the product and measuring the resulting cellular and humoral immune and/or therapeutic responses, prior to administration.
  • this amount represents approxi ⁇ mately 10 4 to about 10 7 plaque forming units, preferably about 1 X 10 6 to about 5 X 10 6 plaque-forming units (determined by assay, as described herein) . It is noted that the above described range of administered vaccinia virus is chosen to enhance the likelihood of eliciting an immunogenic response without causing a malaria infection in the vaccine recipient.
  • the amount of chimeric peptide administered will be an amount or concentration of the peptide to produce a protective immune or therapeutic response with respect to the disease malaria. While this amount may vary over a considerably wide range, depending upon the immunogenicity of the chimeric peptide chosen, generally the amount of peptide administered ranges from about 0.01 micro- grams (10 nanograms) to about 250 micrograms, more preferably about 0.1 microgram to about 100 micrograms, even more preferably about 1 microgram to about 25 micrograms within this range.
  • the present invention contemplates, as a preferred embodiment, the incorporation of both a signal peptide and anchor peptide along with the MSAl peptide into the expressed peptide has been found to be particularly advantageous in producing an immunogenic response to the MSAl peptide.
  • the signal peptide sequence is generally incorporated into the immunogenic peptide at or near the amino end of the MSAl pep ⁇ tide or related antigenic peptide and the anchor peptide is generally incorporated at or near the carboxyl end of the MSAl peptide.
  • the immunogenic peptide is expressed by the vaccinia virus accordingly and will contain a signal peptide aequence and/or an anchor peptide sequence.
  • the signal and anchor peptides are preferably expressed at the amino and carboxy terminus of the expressed MSAl peptide, respectively.
  • the signal peptide sequence is located upstream from the MSAl peptide and the anchor peptide is downstream from the MSAl peptide.
  • any method which is capable of incorporating a sequence of DNA containing genetic material for the expression of the MSAl peptide and optionally, a signal peptide and/or an anchor peptide may be used.
  • the method which is used in the present invention is well known in the art.
  • a DNA sequence containing the genetic code for the MSAl peptide to be expressed is obtained by chemical synthesis or other means such as biochemical isola ⁇ tion of available MSAl DNA sequences and incorporated into a cloning plasmid (for example following cloning vectors: pBR322, pGEM3z, pSP70, pSE420, pRSET, lambdaZAP, all commer ⁇ cially available, among numerous others) .
  • a cloning plasmid for example following cloning vectors: pBR322, pGEM3z, pSP70, pSE420, pRSET, lambdaZAP, all commer ⁇ cially available, among numerous others
  • the appropriate DNA sequence is cloned, isolated, for example, using agarose gel electrophoresis and then incorporated into an amplification vector and amplified by a standard polymerase chain reaction technique for a sufficient number of cycles to obtain a desired quantity of DNA (depending upon the amount of DNA desired, from about 5 cycles to about 40 cycles or more) .
  • a signal peptide sequence and/or anchor peptide sequence may be incorporated into a vector containing the MSAl peptide and, after identification (selection and screening) of the appropriate DNA fragments in positive clones by PCR and endonuclease digestion, amplified accordingly using the same techniques.
  • the DNA is incorporated into a transfer vector and transfected with eukaryotic cells, for example, monkey kidney cells (BSC-l cells) , and with wild-type vaccinia virus (WR) to produce recombinant vaccinia virus.
  • BSC-l cells monkey kidney cells
  • WR wild-type vaccinia virus
  • the recombinant vaccinia virus is then purified before amplification.
  • the recombinant vaccinia virus is then administered to an animal as an immunogenic dosage form which expresses MSAl peptide or an immunogenic portion thereof, preferably, in combination with a signal peptide and/or an anchor peptide.
  • the expression vector may be used for the purpose of expressing the immunogenic chimeric protein in a suitable eukaryotic cell system, for example, to promote the production of the desired peptide sequence outside of the host animal.
  • suitable eukaryotic cell systems include, for example, HeLa, L929, T2 or RMA-2, preferably T2 or RMA-S.
  • the cells which contain the expression vector(s) are grown and then lysed in order to isolate synthetic peptides which con ⁇ tain the desired protein or peptide sequence in combination with the anchor peptide sequence and/or the signal sequence.
  • the isolated peptide sequence may then be used directly as a therapeutic or immunogenic dosage form.
  • the expression vector may be administered directly to the patient where it will express the desired protein or peptide and anchor sequence and render the intended therapeutic or immunogenic effect on the patient.
  • the expressed protein may be obtained from cell cul ⁇ ture after the cells are lysed by standard protein purifica ⁇ tion procedures known in the art which may include, among others, gel electrophesis, affinity and immunoaffinity chromatography, differential precipitation, molecular sieve chromatography, isoelectric focusing and ion-exchange chromatography.
  • standard protein purifica ⁇ tion procedures known in the art which may include, among others, gel electrophesis, affinity and immunoaffinity chromatography, differential precipitation, molecular sieve chromatography, isoelectric focusing and ion-exchange chromatography.
  • immunoaffinity chromatography the protein or peptide may be purified by passage through a column containing a resin to which is bound antibodies which are specific for at least a portion of the protein or peptide.
  • the expressed protein or peptide containing a signal peptide sequence and/or an anchor peptide sequence may be administered in pure or substantially pure form to a patient in need of such therapy by purifying the crude lysate from cell culture.
  • the expressed protein is administered in pharmaceutical dosage form as a composition or formulation comprising an immunogeni- cally effective amount of the expressed protein containing anchor peptide sequence and/or signal peptide sequence, in combination with a pharmaceutically acceptable additive, car ⁇ rier or excipient.
  • the formulations may be delivered in unit dosage form prepared by known methods in the art.
  • the amount of expressed protein or peptide administered will vary depend ⁇ ing upon the pharmokinetic parameters, severity of the disease treated or immunogenic response desired.
  • dosages will be set by the prescribing physician considering relevant factors including the age, weight and condition of the patient including, in the case of immunogenic dosage forms, whether the patient has been previously exposed to the microorganism responsible for the disease to be vaccinated against as well as the release characteristics of the expressed protein from pharmaceutical dosage forms of the present invention.
  • the amount of the expressed protein which is administered according to the present invention comprises an amount effective to produce the intended effect, i.e., to obtain an immunogenic response in the patient which provides a substantially protective effect against malaria.
  • the vaccine which is administered according to the present invention comprises an amount of an expression vector, preferably, a recombinant vac ⁇ cinia virus effective to express sufficient MSAl peptide to provide an immunogenic response in a patient.
  • the MSAl peptide or an immunogenic peptide sequence thereof is combined with a signal and/or anchor peptide to substantially increase the immunogenicity of the expressed MSAl peptide com ⁇ pared to MSAl peptide which does not contain a signal and/or anchor peptide.
  • the immunogenic response provides a pro ⁇ tective effect against the merozoite stage of malaria.
  • the present vaccine can be injected as is, or for con ⁇ venience of administration, can be added to a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable car ⁇ riers will be apparent to those skilled in the art, and include water and other polar substances, including lower molecular weight alkanols, polyalkanols such as ethylene glycol, polyethylene glycol, and propylene glycol as well as non-polar carriers.
  • Dosages of recombinant vaccinia virus or chimeric protein or peptide according to the present invention which are coadministered with carriers will often be about the same as the amount administered alone (in the absence of coad- inistration) .
  • dosages will be set by the pres ⁇ cribing physician considering relevant factors including the age, weight and condition of the patient including whether the patient has been previously exposed to Plasmodium falciparum and the release characteristics of the vaccinia virus from pharmaceutical dosage forms of the present invention.
  • the dose of vaccinia virus will depend upon the form in which it is administered.
  • the vaccine will generally contain a concentration of virus ranging from about 10 4 to about 10 7 plaque forming units, preferably about 1 X 10 6 to about 5 X 10 6 plaque-forming units, depending upon the desired levels of expressed immunogenic protein.
  • concentration or amount of vaccinia virus included within the present vaccine will generally fall within this range; however, the amount of recombinant vaccinia virus used in any vaccine form will depend upon the strength of the immunogenic response elicited.
  • vaccinia virus in a given vaccine dose, the following method may be used.
  • standard pharmaceutical carriers as described above may be included.
  • the ratio of virus included in the vaccine will depend on the chemical nature, solubility, and stability of the virus, as well as the dosage con ⁇ templated.
  • parenteral administration or injection via such parenteral routes as intraperitoneal, intramuscular, sub ⁇ cutaneous, intramammary or other route
  • sterile solutions of the vaccinia virus are prepared.
  • Vaccines according to the present invention may also be administered intravenously.
  • the vaccines according to the present invention are administered via a subcutaneous route.
  • the dosage of the vaccine employed and the treatment schedule would follow practices normally employed for other vaccination or therapeutic regimens wherein this general method of treatment is employed. It is not anticipated that more than one dose of vaccine initially would be required, but the possibility of providing booster doses is anticipated.
  • the dosage schedule for immunization against malaria involves the subcutaneous injection of at least about 1 X 10 6 plaque-forming units of vaccinia virus.
  • a human patient is administered with an effective amount of vaccinia virus such that expressing the MSAl peptide or an immunogenic peptide thereof, preferably in combination with a signal and/or anchor peptide.
  • an immunologically effective chimeric peptide comprising the MSAl peptide or an immunogenic portion thereof in combination with a signal peptide and/or anchor peptide will be admnistered.
  • an additional boost of vaccinia virus or peptide may be given to promote the immunogenic response. Additional doses of vaccine may be provided to boost the ini ⁇ tial inoculation, if needed.
  • D-MEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Wild type vaccinia virus Western Reserve (WR) was grown in BSC-l cells.
  • P. falciparum isolates (FCR-3, a strain identical to the Wellcome strain sequenced by Holder, et al., Nature, 1985, 317, 270-273) were maintained in human erythrocytes in RPMI 1640 medium supplemented with 25 mM Hepes, 32mM sodium bicar ⁇ bonate and 10% human serum in a 5% O 2 , 5% CO 2 , 90% N 2 environ ⁇ ment at 37°C.
  • P. falciparum strain FCR-3 was kindly donated by Dr. Isabella Quakyi, Georgetown University, Washington, D.C.
  • pME-2 is an expression plasmid derived from pWRL507, in which the C-terminal of the MSAl gene (3555-5917bp, Wellcome Allele of MSAl) was inserted at the 3' end of a trun ⁇ cated trpE gene, so that expression was controlled by the trp promoter.
  • the protein was expressed using the method described by Holder, et al., Paraite Immunol.. 1988, 10, 607. The fusion protein expressed was insoluble.
  • DH5alpha competent cells (GIBCO BRL) were transformed by pME-2, and a 1ml culture of a pME-2 positive clone was innoculated into 100ml M9 medium containing 100 ug/ml (micrograms per ml) Amp and grown over ⁇ night. This overnight culture was added to 400ml M9 containing 100 ug/ml Amp and 10 ug/ml indoleacrylic acid, and after 5 hr. growth at 37°C and 250 rpm, the cells were harvested. The pel ⁇ let was then rinsed in 10 ml PBS and frozen to -20°C. The cell pellet was thawed in a 10ml solution contiaining 25 mM Tris pH 8.0, ImM EDTA, 0.2% NP-40 and 100 ul lOOmM PMSF.
  • the material was centrifuged as above to yield a futher pellet and this pellet was resuspended in a 10 ml solution of 0.5M KSCN, 50mM tris, 5mM EDTA and 5mM EGTA and re-centrifuged as above. Finally, the pellet was resuspended in 3 ml water.
  • the product was anayzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) , and the gel containing the 105 kD protein of interest was then cut into approximately 1-5 mm 2 pieces.
  • the fusion protein was eluted with a Bio-Rad Model 422 Electro-Eluter, and the vacuum-dried product was dissolved in PBS.
  • the concentration was assayed with a BCA protein Assay Reagent Kit (PIERCE) .
  • Antiserum to the fusion protein was prepared by immunizing Balb/c mice intraperitoneally on two occasions 21 days apart with 100 ug of protein in the presence of Titre-Max (Vaxcel, Inc.) . Antiserum was collected 10 days after the second immunization.
  • PGEX-2T-P190CR1 (pGEX-2T containing the MSAl gene from 1 to 150bp) was used as a sample in PCR.
  • the lOOul mixture contained 100 pmol primer 1 and 100 pmol primer 2 (Table 1, below), 2.5units Ampli Taq DNA polymerase, dNTP and lOul lOx reaction buffer (PIERCE) .
  • the sample was overlaid with several drops of mineral oil to prevent evaporation and sub ⁇ jected to 30 cycles of amplification (94°C melt, 72°C exten ⁇ sion, 55°C annealling) . Amplified products were identified on a 1.5% agarose gel.
  • Fragment 418-582 1 GC GTCGAC ATG AAG ATC ATA TTC TTT TTA
  • MSAlC-(Si,nA) , MSA1C-(nSi,nA) , MSAlC-(nSi,A) fragments contain the 108bp region directly downstream from the signal sequence and an additional 2bp on the 5' end of the C-terminal to preserve the reading frame. Furthermore, a start codon was also added to the two fragments lacking the start-codon- containing signal sequence, a stop codon was also added to the two fragments lacking the anchor region.
  • the MSA1C-A fragment was amplified by PCR, and elec ⁇ trophoresis in a 0.8% agarose gel indicated that the amplified fragment was about 1.8kb.
  • This fragment and pSPORTl were digested with EcoRI and Kpnl and were mixed and treated with T4 DNA ligase. The ligation products were transformed into DHSalpha competent cells. X-gal and ampicillin were used to screen the positive clones (pSPORTl-MSAlC-A, Figure 1) .
  • Recom ⁇ binant plasmids were prepared from positive white colonies and identification was performed with Sail and EcoRI digestion. The full sequence of the insert was determined.
  • the fragment containing the signal sequence was amplified by PCR, and electrophoresis in a 1.5% agarose gel indicated that the size of the amplified fragment was about 180bp.
  • the Si and pSPORTl-MSAlC-A fragments were then digested with Sail and EcoRI, ligated and transformed, and 30 colonies were selected and screened by PCR using primer 1 and primer 2.
  • Recombinant plasmids (pSPORTl-MSAlC-(Si,A) , Figure 1) were identified with Sail and EcoRI digestion and were sequenced, and the correct reading frame was established.
  • pSPORTl-MSAlC- (Si,A) was then used as a template to amplify MSA1C-(Si,nA) , MSAlC-(nSi,A) and MSA1C-(nSi,nA) .
  • Figure 1 diagrams the construction of recombinant vac ⁇ cinia viruses incorporating the sequences corresponding to MSAlC-(Si,A) , MSAlC-(Si,nA) , MSA1C-(nSi,A) and MSA1C-(nSi,nA) .
  • the MSAlC-terminal fragment containing the anchor region was inserted into the pSPORTl plasmid using the marked EcoRI and Kpnl sites, creating the pSPORTl-MSAlC-A plasmid.
  • the fragment containing the signal region (FSi) and the 108 bp downstream was PCR amplified with a SALIU site on the 5' end and then inserted into the pSPORTl-MSAlC-A plasmid to produce pSPORTl-,SAlC-(Si,A) plasmid.
  • the entire MSAlC-(Si,A) frag ⁇ ment was then removed and inserted into the pSC65 vector to make the final pSC65- MSAlC-(Si,A) transfer vector.
  • the other three recombinant transfer vectors (pSC65-MSAlC-(nSi,nA) , pSC65-MSAlC-(nSi,A) and pSC65-MSAlC-(Si,nA) ) were produced by adding the desired Sail or Kpnl sites where necessary by PCR amplification. The amplified fragments were then inserted into the pSC65 plasmid. In all four cases, the insertion site is adjacent to the synthetic early/late promoter (pS-E/L) . kL and TKp, right and left regions of the vaccinia virus thymidine kinase gene; LacZ, beta-galactosidase gene.
  • Figures 2-5 are the DNA (gene) sequences for MSA1C -(Si,A), MSAlC-(Si,nA) , MSA1C-(nSi,A) and MSAlC- (nSi,nA) .
  • Monolayers of BSC-l cells were grown to 90% confluence in a six-well plate, media was removed, and the cells were infected with wild-type vaccinia virus (WR) at 0.1-1 plaque forming units(pfu) /cell for 1 hour. The virus inoculum was removed, and the monolayer was washed twice with OptiMEM (GIBCO BRL) serum-free medium.
  • WR wild-type vaccinia virus
  • OptiMEM OptiMEM
  • Opti-MEM 1ml of Opti-MEM was added to the infected monolayers and mixed gently with 50 ul of lipofectin-DNA complex (5 ug of recombinant pSC65 was diluted to 25 ul with sterile distilled water, 15 ug of lipofectin reagent was diluted to 25 ul with sterile distilled water, and the solution was gently mixed in a polystyrene tube and allowed to stand for 15 minutes at room temperature) . After 5 hours incubation at 37°C, the medium was replaced with 3 ml E- MEM supplemented with 2% FBS and incubated for another 48 hours. After removal of the medium, cells were harvested by scraping into 1 ml of E-MEM supplemented with 2% FBS.
  • the virus was released by three cycles of freeze-thawing at 37°C. After removal of medium, 1ml of diluted freeze-thawed trans- fection mixture (sonication 30 seconds at 4°C before adding) was added to monolayers of Hu 134 TK- grown to 90% confluence in a six-well plate, and the virus was left for 1 hour at 37°C.
  • the infected cells were overlaid with E-MEM supplemented with 2% FBS containing 1% low-melting-point agarose and 25 ug/ml of bromodeoxyuridine.
  • BSC-l cells were infected with recombinant vaccinia virus, and the medium was removed from infected tissue-culture plates 24 hours postinfection.
  • the cells were fixed with a 1:1 acetone:methanol mixture for 2 minutes, the wells were washed with lml of PBS, and then anti-MSAlC-A serum diluted 1:200 in PBS containing 2% FBS was added to the wells, 1 ml/well.
  • the six-well plate was incubated at room temperature for 1 hour, rocking gently, after which the wells were washed twice with 1 ml of PBS.
  • Anti- ouse-peroxidase diluted 1:1000 in PBS with 2% FBS was added to each well, and the plate was incubated for 45 minutes at room temperature. After washing twice with PBS, 0.5 ml of substrate solution was added (the substrate solution was made by dissolving a pinch of dianisidine in 500ul of absolute ethanol, vortexing, and warming it for 5-10 minutes, then centrifuging it for 30 seconds and adding 200ul of sub ⁇ strate solution to 10 ml of PBS plus lOul of 30% H 0 2 ) . The plate was then left for 5-10 minutes at room temperature.
  • HeLa cells were seeded on pre-treated coverslips for 48h, after which the cells were infected at an M.O.I, of 5 in a volume of 0.25 ml of D-MEM uith 10% FBS. These were over ⁇ laid with 1.5 ml D-MEM with 10% FBS 1-2 h postinfection. The cells were then washed with PBS and fixed for 15 min in 3% paraformaldehyde in PBS. After being washed in PBS, the coverslips were incubated in 50mM ammonium chloride in PBS for 10 min at room temperature.
  • Confluent six-well plates of BSC-l cells were infected with recombinant vaccinia virus in 1 ml medium at an M.O.I of 5. Cells from each well were harvested 24 hours post- infection. After centrifugation at 8000 rpm for 5 min, the supernatant was concentrated to 10-15 ul with a microcon 30 (Amicon, Inc.), and the cell pellet was resuspended in PBS to a final volume of 200 ul.
  • the blot was blocked with 5% BSA in Tris-HCl (200mM, 0.85%) for 2 h at room temperature before addition of a 1:500 dilution of anti-MSAlC- A serum. After 2 hours incubation the blot was washed three times in Tris-HCl, pH 7.4 for 10 min per wash. A 1:7500 dilu ⁇ tion in Tris-HCl, pH 7.4 of alkaline phosphatase-conjugated goat-anti-mouse IgG (Promega) was added, and the blot was incubated for 90 min at room temperature before being washed four times in Tris-HCl, pH 7.4 for 10 min each wash. Western blue stabilized substrate for alkaline phosphatase (Promega) was added, and after about 5 minutes, the blot was washed with water to stop development.
  • HeLa S3 cells in E-MEM supplemented with 2% FBS were incubated in a 162 cm 2 flask at 37°C for 18 h, after which the cells were infected with small stock viruses at 37°C for 48 h and pelleted by centrifugation for 5 min at 1800 g.
  • the infected cells were resuspended in 10 mM Tris-HCl, pH 9.0 and homogenized with 30-40 strokes (while still on ice) .
  • the mixture was centrifuged for 5 min at 300g to remove nuclei, and the pellet (removed supernatant was kept was well) was resuspended in 10 mM Tris-HCl, pH 9.0 and centrifuged again (supematants were pooled) .
  • the sonicated supernatant was layered onto a cushion of 36% sucrose in a Beckman SW27 centrifuge tube and spun at 35000 rpm for 45 min at 4°C, after which the virus pellet was resuspended in 1 ml of ImM Tris- HCl, pH 9.0.
  • the wild-type virus was further purified by con ⁇ tinuous sucrose gradient centrifugation.
  • Monolayers of BSA-1 cells were grown to 95% confluence in a six-well plate, virus stocks were sonicated, and 10 fold serial dilutions(10 ⁇ 7 -10 ⁇ 9 ) of the virus in E-MEM supplemented with 2% FBS were made.
  • the BSC-l cells were infected with 1 ml of virus diluted to 10 ⁇ 7 ,10 ⁇ 8 and 10 ⁇ 9 in duplicate. After 48 h of incubation, the medium was removed and 0.5 ml of crystal violet solution was added. After another 15 min incubation at room temperature, the plates were washed with water, and the plaques were counted after drying.
  • Purified WR vaccinia virus was diluted in Tris-NaCl, pH 7.4 to a final concentration of approximately 5X10 6 pfu/ml and 100 ul volumes were dispensed into the wells of a 96-well plate (IMMULON 2, Dynatech Laboratories Inc) and left for 2 h at 37°C. The buffer was removed and virus was inactivated with 50 ul of 10% paraformaldehyde for 10 min at 4°C. The plates were then blocked just as those in the first group and developed using the procedure described above.
  • P. falciparum parasites were synchronized twice with 5% sorbitol (Diana) , and after 24 hours a mixture of trophozoites and schizonts at 3% parasitemia and 3% hematocrit was transfered to 96-well culture plates (170ul/well) .
  • Anti ⁇ serum, pre-immune serum or serum-free medium was added to the parasites in a volume of 30 ul to make a final volume of 200ul/well, and the plates were incubated at 37°C in an air ⁇ tight chamber equilibrated for 5 min with 5% 0 2 , 5% C0 2 and 90% N 2 . After 22-24 hours, cell morphology was verified to make sure that all schizonts had burst.
  • T is the parasitemia of tested serum
  • pre is the parasitemia of preimmune serum
  • 0 hr is the starting par ⁇ asitemia
  • pSC65-MSAlC-(Si,A) which is recom ⁇ binant pSC65 in which the MSAl peptide containing the signal and anchor regions of MSAl has been inserted into the Sail and Kpnl site;
  • pSC65-MSAlC-(Si,nA) the same except the MSAl con ⁇ tains the signal without the anchor;
  • pSC65-MSAlC-(nSi,A) the same except the MSAl contains the anchor without the signal
  • pSC65-MSAlC-(nSi,nA) the same except the MSAl contains neither the signal nor the anchor
  • the vaccinia virus transfer vector, pSC65 has a synthetic compound early/late promotor (pSE/L) so that the foreign genes controlled by the promotor are expressed throughout the virus growth cycle.
  • the Sail, Bglll, stul, AatI, Kpnl, Smal, Xmal and Pad sites are located just downstream of the pSE/L for insertion of a foreign gene, and there are E.
  • coli beta-galactosidase gene sequences (con ⁇ trolled by the p7.5 vaccinia virus promotor which has early and late vaccinia virus transcriptional regulatory signals) and vaccinia TK gene sequences flanking the entire pSE/ L and foreign gene region to direct homologous recombinantion into the TK locus of the vaccinia virus genome.
  • BSC-l cells were infected with wild-type (WR) vaccinia virus and then were transfected with recombinant pSC65. Serial dilutions of progeny virus were then applied to monolayers of Hu 134 TK- cells in the presence of BrdU to select TK- recom ⁇ binant virus plaques. These were then distinguished from spontaneous TK- mutants by addition of X-gal to the low- melting-point agar overlay. Plaques that stained blue due to expression of beta-galactosidase were picked and then plaque- purified a second time prior to preparation of virus stocks.
  • the four recombinant vaccinia viruses were named as rV.V- MSAlC(Si,A); rV.V-MSA1C-(Si,nA) ; rV.V-MSA1C-(nSi,A) and rV.V- MSAlC(nSi,nA) ( Figure 6).
  • MSA1C- (Si.A) Expression of MSA1C- (Si.A) .
  • MSA1C-fSi,nA) MSA1C- (nSi.A) and MSAlC-fnSi.nA)
  • BSC-l cells were infected with rV.V-N,ISAlC-(Si,A) , rV.V-MSAlC(Si,nA) , rV.V-MSAlC-(nSi,A) and rV.V-MSA1C-(nSi,nA) , and the infected cells were fixed with acetone/methanol 24 hours post-infection, after which the expressed proteins were labeled by anti-mouse-peroxidase. The results indicated that the cells infected by the recombinant vaccinia virus expressed the C-terminal protein.
  • BSC-l cells were infected with rV.V-MSAlC-(Si,A) , rV.V-MSAlC(Si,nA) , rV.V-MSAlC-(nSi,A) , rV.V-MSAIC-(nSi,nA) and WR viruses. These cells were then harvested 24 hours post- infection, and the cell pellets and 50-times concentrated supematants were run on an 8% Tris-glycine-SDS gel. The blot was labled with anti-MSAlC-A mouse serum and then probed by alkaline phosphatase-conjugated goat anti-mouse IgG.
  • the ELISA titers of rV.V-MSAIC-(Si,A) and rV.VMSA1C-(Si,nA) were 5-10 times greater than that of rV.V-MSAIC-(nSi,nA) and rV.V-MSAlC- (nSi,A) after the fourth inoculation.
  • One rabbit was inocu ⁇ lated intravenously on day 0 with rV.V-MSAlC-(Si,A) and reinoculated on days 47 and 68, and the ELISA titer of i.v. rV.V-MSAlC-(Si,A) was 5 times that of i.d.
  • the ELISA titer of i.v. rV.V-MSAlC-SiA decreased sud ⁇ denly after the third inoculation, possibly because the antibody neutralized the virus.
  • rV.V-MSAlC-(Si,A) and rV.V-MSAlC-(Si,nA) stimulated a level of the C-terminal- specific antibodies that was 5-10-fold greater than the level induced by rV.V-MSAlC-(nSi,nA) and rV.V-MSAlC-(nSi,A) after the third inoculation.
  • the ELISA titer of rV.V-MSAlC-(Si,A) was about 1:10000 and lasted about 3 months ( Figure 10).
  • Wild type (WR) vaccinia virus coated on a 96-well plate was probed with a serial 10-fold dilution of mouse antibodies raised against rV.V-MSAIC-(Si,A) , rV.V-MSAlC- (Si,nA), rV.V-MSAlC-(nSi,nA) , rV.V-MSAIC(nSi,A) and WR, and then probed by alkaline phosphatase-labeled goat antimouse IgG.
  • the results showed that the anti-WR virus antibody titers of the recombinant and the WR viruses were almost the same after the second inoculation.
  • mice immunized with the recombinant and WR viruses were successfully infected by the viruses ( Figure 10) .
  • CBA/J mice were also immunized with rV.V-MSAIC-(Si,A) , rV.V-MSAlC- (Si,nA), rV.V-MSAlC-(nSi,nA) , rV.V-MSAIC-(nSi,A) and WR viruses.
  • the resulting antibody titers were similar to those of the Balb/c mice although slightly lower (data not shown) .
  • Schizont stage parasites resuspended in lx sample buffer were boiled for 5 minutes and loaded onto a 4-20% Tris- glycine gradient gel, and the proteins were transferred to a PVDF membrane by electrophoresis.
  • the blots were then probed by anti-MSAlC-(Si,A) and anti-MSAlC-A and labeled by alkaline phosphatase-conjugated goat anti-mouse IgG.
  • the 190kD protein was recognized by anti-MSAlC-A in a Western blot, so we have concluded that the 190kD protein is MSAl.
  • Anti-MSAIC-(Si,A) recognized the 190kD protein (MSAl) as well.
  • Figure 11 The data demonstrate therefore that the recombinant vaccinia virus correctly expresses the MSAl fragment in mice.
  • the C-terminal region of the MSAl protein was inserted into the vaccinia virus to investigate the immune response produced by this antigen.
  • Four variations of the MSAl fragment were used to create constructs which contained neither, one or both of the original MSAl N-terminal signal sequence and the original C-terminal anchor sequence.
  • the apparent molecular weight of the MSAIC-(Si,A) construct is less than expected and may be due to proteolytic cleavage of the protein during export.
  • the two constructs with the signal sequence are expressed on the cell surface, whereas the two constructs without the signal sequence are not. These data indicate that the signal sequence is necessary for proper export of the expressed recombinant protein to the cell surface. Further ⁇ more, the data show that the signal region alone is sufficient for cell surface expression. It is possible that once the protein has entered the secretory pathway, because of the sig ⁇ nal sequence, there is resultant cell surface expression. The strength and duration of this cell surface expression appears to be greater when the protein contains the anchor region in addition to the signal region and this may be due to hydrophobic interactions. Data indicate also that the protein is not secreted externally and thus presumably remains bound to the cell surface. However, the construct with only the signal sequence elicits a weaker immune response than does the construct with both the signal and anchor regions. The anchor region may be necessary for proper protein conformation which is necessary for a high immune response.
  • Immunofluoresence data illustrate that antibodies induced by the MSAlC-(Si,nA) and MSAlC-(Si,A) constructs recognize the parasite in vitro. This indicates that the recombinant protein expressed by the vaccinia virus is indeed very similar, if not identical, to the native parasite MSAl C- terminus. Antibodies targeting the vaccinia viruses indicate that all four recombinant virus constructs as well as WR were administered at approximately the same level. Therefore the differences of the antibody responses of these four constructs is due to the primary sequence of the protein, not varying levels of vaccinia virus.
  • the recombinant protein containing both the signal and anchor regions elicits the greatest immune response and this suggests that both the signal and anchor sequences are advantageous for optimal expression of the MSA-1 C-terminal region.
  • antibodies to the protein containing both the signal and anchor sequences most effec ⁇ tively inhibit invasion of erythorcytes by parasites. Although 62% inhibition of invasion may seem only a partial inhibition, these invasion assays were conducted with a 3% starting parasitemia, whereas most other studies are done at approximately 0.3%. A higher starting parasitemia may reduce the inhibition because of the large numbers of merozoites released.
  • MSAlC-(Si,A) construct expresses a functional protein which possesses the proper sig ⁇ nal sequence and the proper anchor sequence for correct cell surface expression. Furthermore, the antibodies produced by mice and rabbits immunized uith the recombinant vaccinia virus are able to block parasite invasion in vitro.

Abstract

L'invention concerne un peptide chimère de la malaria, constitué d'un système vaccinal viral qui exprime une protéine correspondant sensiblement à l'antigène majeur 1 de surface de mérozoïte (MSA1) de Plasmodium falciparum ou à une portion immunogène de celui-ci. Dans des formes d'exécution préférées, le peptide de MSA1 est un peptide à 593 aminoacides correspondant aux aminoacides 1047 à 1640 de MSA1 et il est combiné à un peptide signal et/ou à un signal d'ancrage. Les peptides chimères, ayant simultanément des séquences signal et d'ancrage associées à des séquences de MSA1, étaient particulièrement efficaces pour provoquer une réponse immune chez un hôte vacciné.
PCT/US1997/001395 1996-01-29 1997-01-29 Vaccins contre la malaria faisant appel a un peptide de msa1 WO1997026911A1 (fr)

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AU22490/97A AU2249097A (en) 1996-01-29 1997-01-29 Malaria vaccine based upon the addition of a msa1 peptide
JP9527100A JP2000504223A (ja) 1996-01-29 1997-01-29 Ｍｓａ１ペブチドの付加に基づいたマラリアワクチン
EP97905652A EP0885012A4 (fr) 1996-01-29 1997-01-29 Vaccins contre la malaria faisant appel a un peptide de msa1
CA002245002A CA2245002A1 (fr) 1996-01-29 1997-01-29 Vaccins contre la malaria faisant appel a un peptide de msa1
US09/117,415 US6551586B1 (en) 1996-01-29 1998-11-27 Malaria vaccine based upon the addition of a MSA1 peptide

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WO2001061032A1 (fr) * 2000-02-17 2001-08-23 Lg Chem Investment, Ltd. Reactif de dosage immunologique et de diagnostic pour le paludisme
WO2001085927A1 (fr) * 2000-05-08 2001-11-15 Georgetown University Vaccin contre la malaria et methode d'utilisation en fonction d'un nouveau domaine antigenique du plasmodium falciparum
KR100427513B1 (ko) * 2000-02-17 2004-04-30 주식회사 엘지생명과학 말라리아 특이 면역글로블린 엠의 검출에 의한 말라리아의면역학적 측정방법, 진단시약 및 진단방법
US7354594B2 (en) 1997-10-20 2008-04-08 Gtc Biotherapeutics, Inc. Merozoite surface protein 1 lacking glycosylation sites

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AUPP589398A0 (en) 1998-09-14 1998-10-08 Walter And Eliza Hall Institute Of Medical Research, The Immunogenic compositions and uses thereof
CN1176945C (zh) * 2001-02-01 2004-11-24 中国人民解放军第二军医大学 疟原虫融合抗原及其制法和用途
EP1527085A2 (fr) * 2002-03-04 2005-05-04 The United States of America as represented by the secretary of the Navy Antigenes et leur utilisation comme agents diagnostiques et vaccins contre les especes de plasmodium
WO2003084472A2 (fr) * 2002-04-01 2003-10-16 Walter Reed Army Institute Of Research Proteine 142 de merozoite anti p. falciparum servant de vaccin recombinant
CN1681529B (zh) 2002-07-26 2012-09-19 沃尔特及伊莱萨霍尔医学研究院 免疫组合物及其诊断和治疗用途
US7931908B2 (en) 2007-02-23 2011-04-26 Philadelphia Health Education Corporation Chimeric MSP-based malaria vaccine
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US9617560B2 (en) 2011-10-05 2017-04-11 Genvec, Inc. Simian (gorilla) adenovirus or adenoviral vectors and methods of use
EP2764012B1 (fr) 2011-10-05 2022-02-23 GenVec, Inc. Vecteurs adénoviraux et procédés d'utilisation associés
EP2809346A1 (fr) * 2012-02-02 2014-12-10 GenVec, Inc. Vaccin contre la malaria à base d'un vecteur adénoviral
US9676824B2 (en) 2012-05-29 2017-06-13 Genvec, Inc. Herpes simplex virus vaccine

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US7354594B2 (en) 1997-10-20 2008-04-08 Gtc Biotherapeutics, Inc. Merozoite surface protein 1 lacking glycosylation sites
US7501553B2 (en) 1997-10-20 2009-03-10 Gtc Biotherapeutics, Inc. Non-human transgenic mammal comprising a modified MSP-1 nucleic acid
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KR100427513B1 (ko) * 2000-02-17 2004-04-30 주식회사 엘지생명과학 말라리아 특이 면역글로블린 엠의 검출에 의한 말라리아의면역학적 측정방법, 진단시약 및 진단방법
WO2001085927A1 (fr) * 2000-05-08 2001-11-15 Georgetown University Vaccin contre la malaria et methode d'utilisation en fonction d'un nouveau domaine antigenique du plasmodium falciparum

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KR19990082086A (ko) 1999-11-15
AU2249097A (en) 1997-08-20

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